Artigo Acesso aberto Revisado por pares

Calpain Activation by Cooperative Ca2+ Binding at Two Non-EF-hand Sites

2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês

10.1074/jbc.m310460200

ISSN

1083-351X

Autores

Tudor Moldoveanu, Zongchao Jia, Peter L. Davies,

Tópico(s)

Calpain Protease Function and Regulation

Resumo

The active site residues in calpain are mis-aligned in the apo, Ca2+-free form. Alignment for catalysis requires binding of Ca2+ to two non-EF-hand sites, one in each of the core domains I and II. Using domain swap constructs between the protease cores of the μ and m isoforms (which have different Ca2+ requirements) and structural and biochemical characterization of site-directed mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding to this domain as shown by the crystal structure of D106A μI-II. However, at elevated Ca2+ concentrations this mutant still forms the double salt bridge that links the two Ca2+ sites and becomes nearly as active as μI-II. Elimination of the bridge in E333A μI-II has a more drastic effect on enzyme action, especially at low Ca2+ concentrations. Domain II Ca2+ binding appears essential, because Ca2+-coordinating side-chain mutants E302R and D333A have severely impaired μI-II activation and activity. The introduction of mutations into the whole heterodimeric enzyme that eliminate the salt bridge or Ca2+ binding to domain II produce similar phenotypes, suggesting that the protease core Ca2+ switch is crucial and cannot be overridden by Ca2+ binding to other domains. The active site residues in calpain are mis-aligned in the apo, Ca2+-free form. Alignment for catalysis requires binding of Ca2+ to two non-EF-hand sites, one in each of the core domains I and II. Using domain swap constructs between the protease cores of the μ and m isoforms (which have different Ca2+ requirements) and structural and biochemical characterization of site-directed mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding to this domain as shown by the crystal structure of D106A μI-II. However, at elevated Ca2+ concentrations this mutant still forms the double salt bridge that links the two Ca2+ sites and becomes nearly as active as μI-II. Elimination of the bridge in E333A μI-II has a more drastic effect on enzyme action, especially at low Ca2+ concentrations. Domain II Ca2+ binding appears essential, because Ca2+-coordinating side-chain mutants E302R and D333A have severely impaired μI-II activation and activity. The introduction of mutations into the whole heterodimeric enzyme that eliminate the salt bridge or Ca2+ binding to domain II produce similar phenotypes, suggesting that the protease core Ca2+ switch is crucial and cannot be overridden by Ca2+ binding to other domains. Calpains are cytosolic cysteine proteases that show a strict Ca2+ requirement for activity (1Lid S.E. Gruis D. Jung R. Lorentzen J.A. Ananiev E. Chamberlin M. 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Calpains have been found in some prokaryotes (Porphyromonas gingivalis) and single cell eukaryotes (Saccharomyces cerevisiae) but are better characterized in multicellular organisms like C. elegans, Drosophila melanogaster, and humans (2Sorimachi H. Suzuki K. J. Biochem. 2001; 129: 653-664Crossref PubMed Scopus (246) Google Scholar). They have in common a cysteine protease core region composed of two structural domains, I-II (∼330 residues). Most calpains have additional C-terminal domains, the two most common being a C2-like domain and a Ca2+-binding penta EF-hand (PEF) 1The abbreviations used are: PEFpenta EF-handDSCdifferential scanning calorimetryHSQCheteronuclear single quantum coherenceIWFintrinsic tryptophan fluorescenceSLY-MCAsuccinyl-leucine-tyrosine-aminomethyl coumarinMES4-morpholineethanesulfonic acidwtwild typer.m.s.d.root mean square devation.1The abbreviations used are: PEFpenta EF-handDSCdifferential scanning calorimetryHSQCheteronuclear single quantum coherenceIWFintrinsic tryptophan fluorescenceSLY-MCAsuccinyl-leucine-tyrosine-aminomethyl coumarinMES4-morpholineethanesulfonic acidwtwild typer.m.s.d.root mean square devation. domain (18Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 19Maki M. Narayana S.V. Hitomi K. Biochem. J. 1997; 328: 718-720PubMed Google Scholar, 20Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). N-terminal combinations are also observed such as the zinc finger domains of SOL calpains (2Sorimachi H. Suzuki K. J. Biochem. 2001; 129: 653-664Crossref PubMed Scopus (246) Google Scholar). Several calpain isoforms (μ, m, and nCL-4) form heterodimers with the ubiquitously expressed calpain small subunit (ss1), through their homologous C-terminal PEF domains (2Sorimachi H. Suzuki K. J. Biochem. 2001; 129: 653-664Crossref PubMed Scopus (246) Google Scholar). This dimerization was illustrated in the crystal structures of the Ca2+-free m-calpain heterodimer (18Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 21Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar). In addition to its C-terminal PEF domain, the small subunit contains an N-terminal glycine-rich domain V that is too mobile to see in the x-ray crystal structure. Other calpains have been shown to exist without the small subunit, including p94 (calpain 3), the calpain 3 lens-specific alternative transcript Lp82, and the Drosophila calpains A and B (2Sorimachi H. Suzuki K. J. Biochem. 2001; 129: 653-664Crossref PubMed Scopus (246) Google Scholar). penta EF-hand differential scanning calorimetry heteronuclear single quantum coherence intrinsic tryptophan fluorescence succinyl-leucine-tyrosine-aminomethyl coumarin 4-morpholineethanesulfonic acid wild type root mean square devation. penta EF-hand differential scanning calorimetry heteronuclear single quantum coherence intrinsic tryptophan fluorescence succinyl-leucine-tyrosine-aminomethyl coumarin 4-morpholineethanesulfonic acid wild type root mean square devation. Although most calpains are complex multidomain proteins, the nCL-2′ splice variant of nCL-2 (calpain 8) and the P. gingivalis calpain contain only the two protease core domains. Consistent with this observation, we have recently shown that the protease core from μ-calpain (referred to here as μI-II or μ-minicalpain) is a Ca2+-dependent cysteine protease when expressed in isolation (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The crystal structure of the Ca2+-bound μI-II revealed two novel non-EF hand Ca2+ binding sites, one in each domain, that are conserved from C. elegans to humans. Neither Ca2+ ion is directly involved in catalysis, but rather Ca2+ binding to these sites appears to drive the realignment of the active site residues to the positions seen in the related papain family of cysteine proteases. Based on comparison with the protease core in the inactive m-calpain heterodimer structure (18Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 21Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar), major conformational changes must occur upon Ca2+ binding. Each Ca2+ site is postulated to assemble through the rearrangements of flexible loops, their loading permitting the two domains to switch into the active conformation in a concerted manner (Fig. 1). Although the x-ray structures reveal the starting and end points of the conformational change, they do not explain how the transition is effected. Here we elucidate the order of Ca2+ binding to the core sites, the basis for cooperativity between them, and the overall mechanism for realignment of the active site residues. This activation mechanism is central and fundamental to the calpains. Cloning, Expression, and Purification of μ/m-minicalpain Hybrids and μI-II Mutants—The reciprocal μ/m minicalpain hybrid constructs, μI-mII and mI-μII, were generated by swapping μI or mI coding segments into the corresponding region of the previously described pET24d mI-II or μI-II constructs, both of which have a C-terminal His6 tag (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 23Moldoveanu T. Hosfield C. Lim D. Jia Z. Davies P.L. Nat. Struct. Biol. 2003; 10: 371-378Crossref PubMed Scopus (67) Google Scholar). The AgeI unique restriction site, which occurs naturally in μ-calpain at the DI-DII boundary (μ-217FTGG220), was used as the swap site. An AgeI restriction site was engineered by Kunkel mutagenesis into the corresponding location within mI-II without alteration of its amino acid sequence using the primer ctcggcaatgccaccggtgaagtcttcaaag (24Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (633) Google Scholar). Swapping NcoI-AgeI fragments produced the desired mI-μII and μI-mII hybrids. Primers used to generate amino acid replacements within pET24d-μI-II are identified by their targeted amino acid substitution. In each case a diagnostic restriction site (underlined) was introduced. The primers were: D106A, NdeI agctccctggcatatggccgttcgggtgg; W298A, SacII ctccacttcacccgcggggttccgcat; E302R, ApaI gtcactccagggccctttccaccgcacttcacttcaccccag; D331A, NaeI gacctccagaattcgccggcttccatcttgacc; and E333A, NarI aaggacatccagaaggcgccgtcttccatct. Two mutations (a double and a single) within the pET24d-m80k construct were achieved using the primers E320A/D321A, SacII atccagaattctcccgcggcctgccgttctg; and R94G, KpnI ccttggcagatgtcggtaccggtagcacctcc. To perform protein crystallization in the presence of Ca2+ without the risk of autolysis, the inactivating cysteine 115 to serine mutation was introduced in the D106A μI-II construct using C115S, PvuII agccaggagccagctgtcccccagagc. Cloned and mutated inserts were sequenced (Cortec DNA Services Laboratory, Inc.) to confirm their identity and integrity. Expression in Escherichia coli, purification, and storage of the above constructs and their mutants followed the previously described protocols (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Differential Scanning Calorimetry—Correct folding of the minicalpain mutants was verified by differential scanning calorimetry (DSC) using a VP-DSC calorimeter (Microcal) (25Brewer J.M. Biotechnol. Appl. Biochem. 1999; 30: 173-175PubMed Google Scholar). Temperature change was at the rate of 90 °C/h, and scans were recorded from 20 °C to 60 °C. Prior to analysis, samples were dialyzed overnight in 4 liters of buffer containing 50 mm HEPES (pH 7.6), 200 mm NaCl, 1 mm EDTA, and 1 mm dithiothreitol. The dialysis buffer was filtered using a 0.22-μm filter (Millipore) and used to obtain a baseline during several buffer-buffer scans. Protein-buffer scans were performed at a final protein concentration of 0.2 mg/ml. Using Origin 5.1 software, the data was corrected for the buffer transitions and modeled to one thermal transition to obtain the melting temperature. Limited Proteolysis—Limited proteolysis of μI-II and its mutants was performed at 22 °C using l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin or 1-chloro-3-tosylamido-7-amino-2-heptanone-treated chymotrypsin (Sigma). The substrate concentration was 1 mg/ml in a reaction volume of 150 μl or less. Trypsin or chymotrypsin was present at 0.1 mg/ml. To monitor the progress of proteolysis, aliquots at timed intervals were mixed with 5× SDS sample buffer and were analyzed by SDS-PAGE and Coomassie Blue staining. Activity and Intrinsic Tryptophan Fluorescence Assays—The steady-state kinetics of mutants and μ/m minicalpain hybrids were measured under similar conditions to those originally described for μI-II using the peptide, succinyl-leucine-tyrosine-aminomethyl coumarin (SLY-MCA, Sigma), as substrate (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). These conditions were also maintained for the CaCl2 activity titrations, which involved increasing CaCl2 concentrations up to 175 mm for the minicalpains. Intrinsic tryptophan fluorescence (IWF) measurements and data analysis were performed as previously described (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Crystallization and Structure Determination—Crystallization of D106A/C115S μI-II in the presence of Ca2+ was done by the hanging drop, vapor diffusion method under slightly different conditions from those established for wild-type μI-II, and produced crystals of a new space group. The well solution contained 1.5 m NaCl, 0.1 m MES (pH 6.5), and 10 mm CaCl2. The drop size was less than 5 μl and contained an equal volume of well solution and protein solution (12.5 mg/ml). Crystals were cryo-protected by six serial soakings (each for up to 5 min) in stabilization solutions containing increasing 5-30% (v/v) glycerol. Diffraction data were collected at the Cornell High Energy Synchrotron Source beamline F1, which was equipped with a Quantum-4 charge-coupled device detector (Area Detector Systems Corp.) and a liquid nitrogen cryo unit (Oxford Cryosystems). The diffraction data were processed as previously described for μI-II (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The space group was P212121 with one molecule per asymmetric unit (Table I). The structure solution was determined by molecular replacement using AMoRe, and the structure of the Ca2+-bound μI-II (PDB accession code 1KXR) as a model. Refinement and analysis of the structure were performed using the software described for μI-II (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The ribbon diagrams were generated using Molscript (26Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). The electron density diagram was made using Xfit, and all structure figures were rendered in RASTER3D (27McRee D.E. J. Mol. Graph. 1992; 10: 44-46Crossref Google Scholar, 28Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3873) Google Scholar).Table ICrystallographic dataData collection statisticsTotalOuter shellResolution range (Å)50.00—1.851.92—1.85Measured reflections304,165Unique reflections26,6002,212Completeness (%)97.186.9I/σI23.44.0RmergeLaRmergeL=∑|Ij-〈I〉|/∑Ij, where Ij represents individual measurements for any one reflection, and 〈I〉 is the average intensity of the symmetry equivalent reflections. (RmergeS)bRmergeS=∑(Ij-〈I〉)2/∑Ij2. (%)7.8 (6.3)41.2 (31.5)Refined structural modelFree reflections (5% of unique)1,283RcrystcRcryst=∑|Fo-Fc|∑Fo, where Fo and Fc are observed and calculated structure factor amplitudes, respectively. (%)23.9Rfree (%)25.9No. of protein/solvent/Ca2+ atoms (excl. H)2,537/101/1Bond angle r.m.s.d. (°)1.194Bond length r.m.s.d. (Å)0.005Average Bfactor (Å2)30.3a RmergeL=∑|Ij-〈I〉|/∑Ij, where Ij represents individual measurements for any one reflection, and 〈I〉 is the average intensity of the symmetry equivalent reflections.b RmergeS=∑(Ij-〈I〉)2/∑Ij2.c Rcryst=∑|Fo-Fc|∑Fo, where Fo and Fc are observed and calculated structure factor amplitudes, respectively. Open table in a new tab Validation of Mutants—In this study, the Ca2+-dependent mechanism by which the active site of calpain is aligned for catalysis was investigated using domain swaps and site-directed mutagenesis (Fig. 2) (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Where targeted amino acid changes interrupt or alter a specific step in the process, it is essential to establish that the change in activity is due to the loss of a functional group and not due to the mis-folding of the mini-calpain. In general, mis-folded proteins are either poorly produced in E. coli or accumulate in inclusion bodies. All mutants and swaps used here expressed well in E. coli, remained soluble, and gave final yields of >10 mg/4 liters of culture that are comparable to those obtained with μI-II and mI-II. Another indication of correct folding is that the chromatography profiles of mutant and wild-type proteins were comparable (not shown). All the minicalpains produced discrete, sharp peaks that eluted at similar locations in the profiles. To further validate the mutants, their thermal denaturation profiles were measured using DSC. Each minicalpain, mutant and wild type, showed a single thermal transition (Fig. 3A). The values ranged from 47 °C (wild type) to 44 °C (D106A). The latter mutant was the one for which an x-ray structure was determined, which revealed no significant differences in the protein fold. With the thermal transitions for the other mutants falling in between these flanking values, it can be safely assumed that none of them was structurally compromised. Limited proteolysis with chymotrypsin (Fig. 3B) and trypsin (not shown) confirmed this. The digestion products were essentially the same for each minicalpain after 80 or 320 min, the only difference being a slightly faster rate of breakdown for some mutants like E302R and W298A. The latter and E333A were the subject of NMR analysis by comparison of its HSQC spectra with that of the wild-type μI-II (not shown), which revealed no significant perturbations in the fold of the mutant. Experimental Strategy—In conjunction with the domain swaps and site-directed mutagenesis, we have used two specific biochemical features of the calpain protease core to elucidate its mechanism of activation by Ca2+. One is the large change in IWF that accompanies the Ca2+-driven alignment of the active site for catalysis (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). We provide evidence below that this change is, as previously suspected, largely due to the movement of Trp298. The other is the Ca2+-dependent proteolytic activity of the core as measured by SLY-MCA hydrolysis. The gain in enzyme activity for μI-II is biphasic. The first phase is sigmoidal. It reflects the cooperative binding of the two Ca2+ and parallels the IWF change. The second phase is a gradual gain in activity at unphysiologically high Ca2+ levels, which we attribute to general stabilization of the bilobal core. The core lacks the support of the other domains it would have in the whole enzyme. What readily distinguishes the m- and μ-minicalpains as enzymes is the greatly reduced activity of mI-II due to the collapse of a key helix in domain I leading to a rearrangement of hydrophobic core residues (23Moldoveanu T. Hosfield C. Lim D. Jia Z. Davies P.L. Nat. Struct. Biol. 2003; 10: 371-378Crossref PubMed Scopus (67) Google Scholar). Minicalpain Domain Swaps Demonstrate the Order of Ca2+ Binding—The increase in IWF during Ca2+ titration was measured for four minicalpains, native μI-II and mI-II, and the reciprocal domain swaps, mI-μII and μI-mII. All four gave characteristic sigmoidal profiles that fitted well to the Hill equation (Fig. 4A and Table II). Ca2+ binding was cooperative between the two domains as indicated both by the upward curvature of the Scatchard plots (Fig. 4A, inset) and by the Hill coefficients of ∼2. Ca2+ concentrations for half-maximal change ([Ca2+]0.5) were ∼27 and 190 μm for μI-II and mI-II, respectively (Fig. 4A and Table II). These values are remarkably close to the Ca2+ requirement for activation of the intact μ- (5-50 μm) and m-calpains (200-1000 μm). The Ca2+ requirements for the mI-μII and μI-mII swaps were 33 and 104 μm, respectively (Fig. 4A and Table II), intermediate between those of μI-II and mI-II. Even more informative was the shape of the sigmoidal IWF profiles. The μII-containing constructs, μI-II and mI-μII, produced very similar profiles that saturated at ∼200 μm, whereas the mII-containing constructs, mI-II and μI-mII, both reached a plateau at ∼1 mm. Thus, it appears domain II controls the end point of the titration and binds Ca2+ second. The initial part of the transition was very similar for the μII-containing constructs, μI-II and mI-μII, with both proteins beginning their change at <10 μm Ca2+. We, therefore, reasoned that DI from either isoform has high Ca2+ affinity and binds Ca2+ first, with μI being a slightly better binder than mI.Table IIHill equation and steady-state SLY-MCA hydrolysis parameters[Ca2+]ia[Ca2+]i is the half-maximal Ca2+ requirement obtained during intrinsic tryptophan fluorescence titrations.nHbnH is the Hill coefficient.kcatckcat refers to the nanomoles of MCA released per second per micromole of enzyme.Kmkcat/Kmdkcat/Km is the catalytic efficiency of MCA release.μm× 10−5s−1μm× 10−8 μm·s−1μI-II26.5 ± 1.01.842.7 ± 4.7335 ± 6130mI-mII104 ± 331.423.0 ± 0.3403 ± 957mI-μII32.9 ± 3.41.73.6 ± 0.9460 ± 108mI-II190 ± 41.7NDeND, not determined.NDInactiveD106A μI-II144 ± 211.3NDNDNDW298A μI-II15 ± 31.8NDNDNDE302R μI-II119 ± 31.4NDNDInactiveD331A μI-II103 ± 61.31.4 ± 0.5318 ± 2044(33.1 ± 2.5)fThe parameters obtained at 20 mm CaCl2.(304 ± 9)fThe parameters obtained at 20 mm CaCl2.(109)fThe parameters obtained at 20 mm CaCl2.E333A μI-II292 ± 31.8NDNDNDm80k/21k353 ± 95.63,248 ± 60211 ± 965,000R94G m80k/21k1,485 ± 631.92,246 ± 104189 ± 611,860ED320/321AA8,352 ± 6411.31,341 ± 169131 ± 1210,182a [Ca2+]i is the half-maximal Ca2+ requirement obtained during intrinsic tryptophan fluorescence titrations.b nH is the Hill coefficient.c kcat refers to the nanomoles of MCA released per second per micromole of enzyme.d kcat/Km is the catalytic efficiency of MCA release.e ND, not determined.f The parameters obtained at 20 mm CaCl2. Open table in a new tab We further demonstrated the functionality and hybrid nature of the domain swaps by testing their enzyme activity. We have previously shown that μI-II readily hydrolyzes the calpain substrate SLY-MCA (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In contrast, mI-II fails to cleave SLY-MCA because of an intrinsic inactivation mechanism triggered by the collapse of a key α-helix at Gly203 in domain I (23Moldoveanu T. Hosfield C. Lim D. Jia Z. Davies P.L. Nat. Struct. Biol. 2003; 10: 371-378Crossref PubMed Scopus (67) Google Scholar). This occurs in the Ca2+-loaded state and leads to the protrusion of Trp106 into the active site cleft. Consistent with this, the activity of the mI-containing swap, mI-μII, was barely detectable with a kcat of 3.6 ± 0.9 × 10-5 s-1, and a Km of 0.46 ± 0.01 mm (Fig. 4B and Table II). In contrast, the μI-containing swap, μI-mII, where there is an alanine in place of Gly203, was quite active. Its kcat (2.3 ± 0.3 × 10-4 s-1) was about half that of μI-II, and its Km was slightly higher (0.403 ± 0.009 mmversus ∼0.3 mm). As seen with μI-II, the activity of μI-mII (but not that of mI-μII) was augmented at higher ionic strength due to general stabilization of the core. We have also modeled the swaps using the Ca2+-bound μI-II and mI-II structures (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 23Moldoveanu T. Hosfield C. Lim D. Jia Z. Davies P.L. Nat. Struct. Biol. 2003; 10: 371-378Crossref PubMed Scopus (67) Google Scholar). The domain interface is highly conserved between the isoforms (not shown), and there are no obvious structural impediments to the hybrids undergoing activation by the same mechanism that brings the two domains together in μI-II. Several inferences can be made from the domain swap analysis. Regardless of isoform, the domain I site has a very similar high affinity for Ca2+, being occupied prior to domain II. In contrast, the affinity of domain II is isoform-specific being the principal determinant of Ca2+ requirement within the protease core. Trp298 Contributes Most to the Intrinsic Tryptophan Fluorescence Change of μI-II—Ca2+ loading results in a very substantial IWF increase (∼37% in μI-II and ∼28% in mI-II) that shows the cooperativity of the process and makes the conformational change easy to follow. The prime candidate for the major contributor to the IWF increase is Trp298 (22Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Its indole ring moves from the wedge-like position in between domains, where it is exposed to solvent on both sides, into a Ca2+-induced hydrophobic pocket that fully buries one of its sides (Fig. 1). Because the Trp298 residue remains surface-accessible, rather than contributing to a hydrophobic core, its mutagenesis to alanine is structurally feasible. Its position above the domain II catalytic triad residues (His272 and Asn296) matches that seen in other cysteine proteases, where it shields these residues from solvent exposure and thus facilitates catalysis. We made the W298A mutant in the inactive C115S μI-II, because we predicted that activity would in any case be abolished by exposure of the key catalytic residues to solvent. Indeed, a W288Y mutation within the m-calpain heterodimer resulted in a significantly lower activity ( 90% complete at ∼4

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